Method for determining endohydrolases capable of breaking down polysaccharides and reagents for carrying out the method

ABSTRACT

A METHOD FOR DETERMINING ENDOHYDROLASE WHICH BREAKS DOWN POLYSACHARIDE IN AN AQUIOUS SAMPLE WHICH COMPRISES CONTACTING THE SAMPLE WITH A REAGENT CONSISTING OF A WATER-INSOLUBLE BUT HYDROPHILIC, SWELLABLE, ENZYMATICALLY HYDROLYZABLE THREE-DIMENSIONAL NETWORK OF MOLECULES OF EITHER A POLSACCHARIDE OR AN ENZYMATICALLY HYDROLYZABLE DERIVATIVE OF A POLYSACCHARIDE. THE MOLECULES ARE CROSSLINKED WITH BONDS OF A COVALENT NATURE. ALSO THE THREE-DIMENSIONAL NETWORK PRESENTS INDICATABLE SUBSTITUENTS BOUND BY MEANS OF BONDS OF A COVALENT CHARACTER. THE CONTACTING CAUSES A REACTION TO TAKE PLACE BETWEEN THE ENZYME AND THE REAGENT WHEREBY WATER-SOLUBLE FRAGMENTS OF THE REAGENT CONTAINING INDICATABLE SUBSTITUENTS ARE RELEASED. THE UNDISSOLVED REAGENT IS SEPARATED FROM THE LIQUID WITH THE WATER-SOLUBLE FRAGMENTS OF THE REAGENT DISSOLVED THEREIN, AFTER THE ENZYME HAS ACTED UPON THE REAGENT FOR A DETERMINED PERIOD OF TIME. THE INDICATABLE SUBSTITUENTS ARE THEN DETERMINED IN AT LEAST ONE OF THE TWO PHASES CONSISTING OF THE LIQUID PHASE AND THE UNDISSOLVED REAGENT MATERIAL PHASE AS A MEASURE OF THE ENZYME ACTIVITY. ALSO, THE REAGENT FOR DETERMINING ENDOHYDROLASE IN PROVIDED.

United States Patent METHOD FOR DETERMINING ENDOHYDRO- LASES CAPABLE OF BREAKING DOWN POLY- SACCHARIDES AND REAGENTS FOR CARRY- ING OUT THE METHOD Bjiirn G.-A. Ingelman and Miroslav Ceska, Uppsala, Sweden, assignors to Pharmacia AB, Uppsala, Sweden No Drawing. Filed Jan. 14, 1969, Ser. No. 791,177 Claims priority, applic lafitgo/ngweden, Jan. 15, 1968,

Int. Cl. G011]! 31/14 US. Cl. 195103.5 R 42 Claims ABSTRACT OF THE DISCLOSURE A method for determining endohydrolase which breaks down polysaccharide in an aqueous sample which comprises contacting the sample with a reagent consisting of a water-insoluble but hydrophilic, swellable, enzymatically hydrolyzable three-dimensional network of molecules of either a polysaccharide or an enzymatically hydrolyzable derivative of a polysaccharide. The molecules are crosslinked with bonds of a covalent nature. Also the three-dimensional network presents indicatable substituents bound by means of bonds of a covalent character. The contacting causes a reaction to take place between the enzyme and the reagent whereby water-soluble fragments of the reagent containing indicatable substituents are released. The undissolved reagent is separated from the liquid with the water-soluble fragments of the reagent dissolved therein, after the enzyme has acted upon the reagent for a determined period of time. The indicatable substituents are then determined in at least one of the two phases consisting of the liquid phase and the undissolved reagent material phase as a measure of the enzyme activity. Also, the reagent for determining endohydrolase is provided.

It is highly important that so-called endohydrolases capable of breaking down polysaccharides, can be determined in a simple and reliable manner. A particularly important example in this respect is the determination of the enzyme a-amylase which hydrolyses starches and glycogen. Determinations of a-amylase are made in great numbers, e.g within the field of medicine in urine and serum tests for diagnostic purposes.

Routine methods have hitherto been accompanied with considerable disadvantages, and cannot be said to have been entirely satisfactory. The determination of a-amylase is also important within industry. Similarly, it is also important to be able to determine simply and reliably other endohydrolases which hydrolyse other polysaccharides such as dextran.

The present invention is concerned with a simple and reliable method for determining endohydrolases which break down polysaccharides in aqueous samples, and reagents for carrying out the determinations.

The term polysaccharides as used in the following description and claims relates to both native polysaccharides and synthetic polysaccharides which contain the structural characteristics of the native polysaccharides, said characteristics rendering them degradable by the endohydrolase in question. Examples of such polysaccharides are e.g. starches such as amylose or amylopectine or dextrins thereof, dextran, levan, mannan, galactan or derivatives thereof etc. In the hydrolysation process, the endohydrolase breaks up glycosidic bonds in the polysaccharide or in the degradable derivatives thereof.

. The method for making the determination is mainly characterized in that the sample is contacted with a reagent consisting of a water-insoluble, but hydrophilic,

3,676,303 Patented July 11, 1972 ice swellable, enzymatically hydrolysable three-dimensional network of molecules of the polysaccharide or enzymatically hydrolysable derivates thereof, said molecules being cross-linked by bridges with bonds of a covalent nature, in which network there is also present indicatable groups or atoms bound by means of bonds of covalent character, a reaction taking place between the enzyme and the reagent to release water-soluble fragments of the reagent containing indicatable groups or atoms, whereafter, subsequent to the enzyme having acted upon the reagent for a determined length of time, undissolved reagent substance is separated from the liquid with the Water-soluble fragments of the reagent dissolved therein, whereafter the indicatable groups or the atoms in the liquid or in the undissolved reagent material are determined as a measure of enzyme activity.

A reagent for carrying out the determination is mainly characterized in that it consists of a water-insoluble but hydrophilic, swellable, enzymatically hydrolysable threedimensional network of molecules of the polysaccharide or enzymatically hydrolysable derivatives thereof, said molecules being cross-linked by bridges having bonds of a covalent character, in which network there are also indicatable groups or atoms bound by means of bonds of a covalent character.

By the expression indicatable substituents or indicatable groups or atoms as used in the following description and claims is meant colour-producing groups, fluorescent groups or radioactive isotopes or groups provided with radioactive isotopes. Preferably, however, colouringproducing groups are selected for practical reasons, since it is relatively simple to conduct colour measurements, even in routine laboratories. Nevertheless, when a particularly high degree of sensitivity is required it can be expedient to choose fluorescent groups, or preferably radioactive isotopes or groups provided with radioactive isotopes.

The reagent used to eifect the determination can be prepared in a number of different ways. For instance, it is possible with the aid of bifunctional bridge formers tocross-link molecules of the polysaccharide or derivatives thereof which can be hydrolysed by the endohydrolase in question, by means of bonds of a covalent character to a three-dimensional network, whereafter the indicatable groups or the atoms are coupled to the network by bonds of a covalent character. It is also possible to first couple the indicatable groups or the atoms by bonds of a covalent character to molecules of the polysaccharide or enzymatically hydrolysable derivatives thereof, and only then effect the cross-linking with the aid of bifunctional bridge formers. It is also possible to elfect crosslinking and introduction of the indicatable groups in the same stage, e.g. by using bifunctional bridge formers which also contain the indicatable groups or the atoms, e.g. bifunctional reactive colour-producing substances.

There are naturally many types of bifunctional substances which can be used as bifunctional bridge formers. Examples of such bridge formers are diepoxides and corresponding halogen hydrins and diisocyanates (e.g. hexamethylenediisocyanate) and dithioisocyanates. Such bridge formers can, for example, react with hydroxyl groups or amino groups in the polysaccharide or in derivatives thereof which can be hydrolysed by the endohydrolase, the molecules thereof being linked together by bridges having bonds of a covalent character.

Examples of such bifunctional epoxides and halogen hydrins are dichlorohydrin, epichlorohydrin, epibromohydrin, 1,2 ethanedioldiglycide ether, 1,4-butanedioldiglycide ether, glyceroldiglycide ether, bis[2,3-epoxypropyl]ether and l,2,3,4-diepoxybutane. These are able to react, for instance, with hydroxyl groups and amino groups in the presence of an alkaline substance. For instance, when the reaction takes place with the hydroxyl groups in the polysaccharide, the bridge will be of the type --OAO wherein A is a hydroxyl group-containing alkylene bridge optional-1y broken by one or more oxygen atoms. The cross-linked polymer is insoluble but swellable in water.

Examples of A in the above bridge are:

As is evident from the aforegoing there are many different types of bridges between the molecules of the polysaccharide in the reagent that can be selected. In one suitable embodiment the molecules of the polysaccharide are cross-linked by means of aliphatic bridges having bonds of a covalent character, in which bridges the number of carbon atoms is from 2 to 20 (such as from 3 to 20), preferably from 3 to 15, such as from 3 to It is particularly suitable to select bridges which contain hydroxyl group-containing alkylene groups having from 3 to carbon atoms, preferably from 3 to 15 carbon atoms, such as from 3 to 10 carbon atoms, said groups being optionally broken by one or more oxygen atoms. Such hydroxyl group-containing bridges render the network more hydrophilic and swellable in water, and improve the properties of the network.

The indicatable groups or the atoms can be introduced in a number of different ways. For instance, when it is desired to introduce coloured groups it is relatively simple to react with reactive colour-producing substances capable of reacting, for example, with hydroxyl groups or amino groups in the polysaccharide or derivative of polysaccharide, e.g. colour-producing substances reactive to cotton wool or wool being chosen, said substances being attached to the polysaccharide or polysaccharide derivative with bonds of a covalent character. Depending upon the category of use, the most suitable colour is chosen, for example, a blue or red colour. Examples of such reactive colour-producing substances are Cibacronscharlach 2 G (Procion Scarlet H3GS), Cibacronblau 3 G (Procion Blue HBS). The structure formulae of these substances are given in an article by J. Panchartek et al. in Coll. Czech. Chem. Commun, vol. (1960), pp. 2783-2799. The introduction of fluorescent groups can be effected with, for instance, fluorescein derivatives, e.g. fluorescein isothiocyanate. There are also a variety of ways in which radioactive groups or groups containing radioactive isotopes can be introduced. One example is to introduce a group containing a radioactive isotope of iodine, e.g. 1. It is also possible to introduce first a group having no isotope, and then introduce a radioactive isotope into the group. This can, for example, be done by first introducing an allyl group, e.g. with the aid of allyl bromide, and then adding a radioactive iodoisotope to the double bond.

The degree of substitution with regard to indicatable groups or atoms and cross-linking bridges in the polysaccharide molecules is selected so that the quantity of indicatable groups and atoms present is sufiicient for the determination process and so that the three-dimensional network is sufliciently held together. The degree of substitution, however, is not selected to a level which prevents the polysaccharide molecules in the network from being broken up here and there by the endohydrolase in question. A suitable degree of substitution with regard to indicatable groups and cross-linking bridges for the appropriate polysaccharide and endohydrolases in question can be established experimentally by the expert relatively easily.

If the reagent is desired in particle form the gel product obtained in the aforedescribed synthesis of the reagent material can be ground to the appropriate grain size. The synthesis of the reagent gel can also be effected as a pearl polymerization process, by emulsifying the reaction mixture in an inert liquid with which the reaction mixture is immiscible, small spherical particles being obtained directly. The resulting granular material can be screened in a suitable manner, and fractions having appropriate grain size recovered.

An essential condition of the present invention is that the polysaccharide molecules with attached covalent bonded indicatable groups or atoms (e.g. colour-producing groups) are bound together by bridges having bonds of a covalent character. In this way it is impossible for the indicatable groups or atoms to dissolve other than by hydrolysation of the pertinent bonds in the polysaccharide molecules in the network by the endohydrolases. It is also important that although the reagent is insoluble in water it can be easily attacked by the endohydrolase in question; this requirement being provided for by the fact that the reagent consists of a hydrophilic, waterswellable, three-dimensional loose network bonded together by bridges having bonds of a covalent character. The indicatable groups or the atoms, e.g. the easily measured colour-producing groups, enable the determinations to be made with comparative ease. Furthermore, the combination of the aforedescribed features provides a particularly sensitive and reliable determination method which can be effected simply and is extremely suitable for routine work.

When the endohydrolase acts on the reagent, the polysaccharide chains are broken and Water-soluble fragments of the reagent with attached indicatable groups or atoms are released. These fragments are dissolved, and can readily be isolated from the undissolved reagent material, e.g. by simple filtration methods or centrifuging. The determination is suitably effected by contacting the aqueous sample containing the endohydrolase in question with an appropriate quantity of reagent, and permitting said sample to act upon the reagent under agitation for a predetermined length of time at a temperature suitable for the enzyme in question. Of course, the hydrolysation by the enzyme is effected at a pH suitable for the enzyme in question and in appropriate salt environment. When the enzyme has acted on the reagent for the determined length of time the further enzymatic hydrolysation proc-- ess is halted in the conventional manner. For instance, it is possible to prevent further hydrolysation of the reagent by, for instance, heating or cooling the system, changing the pH or by adding some appropriate inhibitor; it also being possible simply to separate the reagent from the sample solution if the time taken is short in relation to the reaction time. Subsequent to separating the reagent from the sample solution, the indicatable groups or the atoms in the liquid or in the undissolved reagent material are determined as a measure of the enzyme activity. Simplest in this respect is that the indicatable groups are colour-producing groups and that the colour is determined in the fluid as a measurement of the enzyme activity.

The reagent is preferably in particulate form, small particles being selected so that a large contact surface is obtained, although the particles should not be so small as to render separation of the undissolved substance and liquid difficult. By suspending the particles in a suitable liquid under agitation it is possible to fill test tubes to the same volume of the obtained suspension, so that the reagent is introduced into each reagent tube in the same quantities to a sufficient degree of accuracy. Errors caused byvariations in the quantity of substance introduced into the test tubes can also be reduced by using an excess quantity of the substrate for the enzyme.

The reagent, for example in particle form, can be mixed with an inert material, for example, in the form of a support substance. It can thus be mixed with paper pulp to form a paper mixed with the reagent, said paper being possible to cut up into pieces suitable for the analysis.

The method is of particularly great importance for determining endohydrolases which break down starches and glycogen, for instance when a large number of u-amylase determinations are made as a matter of routine for medical purposes and within industry. Present routine methods for determining a-amylase cannot be considered fully satisfactory, and hence the method of the invention fulfills a particularly pronounced requirement. The method can also be used for determining other endohydrolases, e. g. for such which break down dextran.

The quantity of indicatable groups or atoms in the water-soluble fragments which are released and dissolved depends upon the enzyme activity in-the sample and on the time during which the enzyme acts upon the reagent as well as other factors important to enzymatic processes, such as temperature and pH, and in certaininstances, e.g. in the case of a-amylase, the salt content of the sample. In accordance with a preferred embodiment of the invention the determination is therefore effected at a temperature and pH value suitable for the enzyme in question, and when applicable also in a suitable salt environment.

A suitable reaction time for the determination to be made can be established so that the quantity of indicatable groups or atoms in the solution is only a function ofthe enzyme activity in the sample. Of course, it is also possible to measure the amount of indicatable groups or atoms in the solution at different time periods, e.g. when a certain quantity of substance has been obtained in the solution. If desired, comparison tables or graphs can be used to convert obtained values to other units for the enzyme activity in question. r

The invention'will now be illustrated by the following examples.

. EXAMPLE 1 50 g. of starch (soluble, proanalysis) were dissolved in 200 ml. of water. 10 m1. of 10 M NaOH solution were added at 20 C., whereupon 4 ml. of 1,4-butanediol-diglycide ether were slowly added dropwise whilst stirring. The reaction mixture was then left to stand without being agi rated, for two days at 20 C. The resulting gel was then ground into small particles and washed with water.

10 g. of Cibacronblau 3 G-A were dissolved in 200 ml. of water, whereafter the gel particles were suspended in the dye solution. 15 g. of NaCl were also added. After two hours 10 ml. of 10 M NaOH solution were added at 20 C. whilst stirring. The reaction mixture was then left to stand without being agitated for 20 hoursat 20 C. The particles were then washed thoroughly with water. The particles were then dried 'and further ground in a ball mill, down to a mean particle size of approximately 40 microns. The particles are insoluble but swellable in water.

EXAMPLE 2 v The same procedure was adopted as in Example 1 but with 3 ml. of 1,4-butanediol-diglycide ether.

EXAMPLE 3 The same procedure was adopted asin Examplel but with 5 ml. of 1,4-butanediol-diglycide ether.

I EXAMPLE 4 The sameprocedure was adopted as in Examplel but with 8 g. of Cibacronblau 3 G-A.

.of reagent.

EXAMPLE 5 The same procedure was adopted as in Example 1 but with 10g. of Cibacronscharlach 2 G instead of Cibacronblau G-A.

' EXAMPLE 6 50 g. of starch (soluble, p.a.) were dissolved in 200 m1. of water. 10 g. of Cibacronblau 3 G-A and 15 g. of NaCl were added at 20 C. When the dye material had dissolved 10 ml. of NaOH were stirred into the system. The reaction mixture was then left to stand for 20 hours at 20 C., whereafter 5 ml. of 1,4-butanediol-glycide ether were added dropwise whilst stirring. The resulting gel was then ground and washed thoroughly with water. The particles were then dried and ground further in a ball mill, down to a mean particle size of 40 microns.

EXAMPLE 7 The same procedure was adopted as that in Example 6 but with 8 g. of Cibacronblau 3 G-A and 4 ml. of 1,4- butanediol-diglycide ether.

EXAMPLE 8 The same procedure was adopted as that in Example 6 but with 10 g. of Cibacronscharlach 2 G instead of Cibacronblau 3 GA.

EXAMPLE 9 The same procedure was adopted as that in Example 1 but this time with dextran with a molecular eight (M of 460,000 instead of starch.

EXAMPLE 10 The same procedure was adopted as that in Example 6, but this time the dextran with a molecular weight M =460,000 instead of starch.

EXAMPLE 11 The reagents for Examples 1 to 8 were tested with aamylase from pancreas, blood plasma, urine, saliva, malt and Bacillus subtilus. In these instances the colour release can suitably be measured at 620 nanometers for the blue colour and 510 nanometers for the red colour. A suitable buffer for use in the determinations is a 0.02 M sodium phosphate buffer having a pH=7.0, which also contains NaCl (e.g. 0.01 M). 0.02% NaN can also be added to the buffer, to prevent the growth of microorganisms. The determination is preferably efiFected so that the desired quantity of reagent is added with a suitable volume of buffer solution in a reagent tube, whereafter the aqueous sample, which contains a-amylase is added. The reagent tube and its contents are shaken in a thermostat at a suitable temperature for the intended period of time, wh'ereafter further enzymatic hydrolysis is prevented by, for example, heating or cooling the system or changing the pH value or by adding appropriate enzyme inhibitors in a known manner, or by separating the particles from the liquid. The colour measuring process is suitably effected on the liquid, which is separated from the particles by centrifuging or filtration.

The colour release from the reagents from Examples 1 to 8 under the influence of the aforementioned ot-amylases was studied at different temperatures, for instance 20, C. to 60 C., at differentpH-values and sodium chloride concentrations, as a function of time and quantity of enzyme. Tests were also made with varying quantities It was established as a result thereof that the determinations are suitably effected in the aforementioned buffer with pH=7.0, at for instance 37 C. (although higher temperatures can be selected, up to 47 C. in some cases). If desired, however, the determinations can also be effected at room temperature (20 C.) or below; although a long reaction time is then required of course. From a practical point of view it was found that in the case of normal routine determinations of u-amylase the conditions should suitably be chosen so that the quantity of reagent is in the order of -10 mg. and the volume of the aforesaid buffer from 1 to 2 ml., while the volume of the enzyme sample should be 0.010.1 m1., although of course other quantities can be chosen.

It was discovered in the tests that the release of colour in the initial stages is an essentially rectilinear function of the time. It was also found that the colour release is a function of the enzyme activity in the sample, a higher degree of colour release after a certain length of time of course corresponding to a higher degree of enzyme activity in the sample, and reliable relationship curves or tables between released colour and enzyme activity can readily be drawn up for the reagent material in question.

It can be mentioned for the purpose of illustrating the sensitivity of the method that 0.04 microgram of crystalline a-amylase from Bacillus subtilis released from 1 mg. of the reagent from Example 6 after 10 minutes at 47 C. a quantity of colour fully sufficient to effect the measuring process. (OD about 1 at the test conditions in question.)

Pure B-amylase, which is an exoenzyme, should not release colour from this reagent. When testing with pure ,G-amylase from Bacillus subtilis no colour release took place. V

Tests carried out with the aforementioned reagent and a number of other reagents prepared with varying amounts of reactive colour-producing substances and 1,4-butanediol-diglycide ether and other bridge formers such as 1,2- ethanediol-diglycide ether and epichlorohydrin under varying conditions showed the utility of said reagents in determining a-amylase. For instance, the reagents described in Examples 1 and 2 were found very favourable with respect to the degree of substitution of colour-producing groups and cross-linking bridges, and were particularly well suited for tit-amylase determinations.

EXAMPLE 12 To illustrate further the release of water-soluble fragments having attached colour-producing groups, as a function of the reaction time, the used reaction times and corresponding CD -values have been presented in the following Table 1. a-amylase from B. subtilis (0.08 microgram/mil) and the reagent from Example 6 (5 mg./ ml.) were used in the test. The temperature was 60 C. The buffer solution was that described in Example 11,

with a pH=7.0.

TABLE 1 Reaction time in minutes: CD m, 2 1.16

As can be seen, the amount of colour in the solution increases with the reaction time.

EXAMPLE 13 In Table 2 below are given the OD -values in a test with a-amylase from B. subtilis; the quantity of enzyme being varied from 0.04 to 0.40 mg./ml. The quantity of reagent (from Example 6) was 7 mg./ ml. The temperature was 47 C. and the reaction time 10 minutes. The buifer solution was the one described in Example 11, having a pH=7 .0.

TABLE 2 Enzyme, ug/ml: CD mm 0.04 t e.. --s..-- 2.27

EXAMPLE 14 The reagents from Examples 9 and 10 and other reagents prepared with varying quantities of bridge formers and reactive colour-producing substances in relation to the dextran polymer were tested with endodextranase from different microorganisms in a manner similar to that used for the a-amylase in Examples 11, 12 and 13. A release of coloured fragments was also obtained in this instance, in a manner corresponding to that when u-amylase was permitted to act upon the a-amylase reagent described above. What we claim:

1. A method for determining endohydrolase capable of hydrolysing polysaccharide, in aqueous samples, which comprises contacting the sample with a reagent consisting of a water-insoluble but hydrophilic, swellable, enzymaltically hydrolysable three-dimensional network of molecules of a member selected from the group consisting of the said polysaccharide and enzymatically hydrolysable derivatives thereof, said molecules being crosslinked by bridges with bonds of a covalent character, said network also presenting indicatable substituents bound by means of bonds of a covalent character, a reaction taking place between the enzyme and the reagent to release water-soluble fragments of said reagent containing indicatable substituents, and subsequent to the enzyme having acted upon the reagent for a determined period of time, separating undissolved reagent substance from the liquid With water-soluble fragments of the reagent dissolved therein, and then determining the indicatable substituents in at least one of the two phases consisting of the liquid phase and the undissolved reagent material phase as a measure of enzyme activity.

2. A method as claimed in claim 1, wherein the reagent is in particle form.

3. A method as claimed in claim 1, wherein the polysaccharide is a member selected from the group consisting of starches and dextrins thereof and enzymatically hydrolysable derivatives thereof.

4. A method as claimed in claim 1, wherein the indicatable substituents are colour-producing groups.

5. A method as claimed in claim 1, wherein the indicatable substituents are fluorescent groups.

6. A method as claimed in claim 1, wherein the indicatable substituents are each a member selected from the groupconsisting of radioactive isotopes and groups provided with radioactive isotopes.

7. A method as claimed in claim 1, wherein the determination is eifected in the presence of a butter having an appropriate pH for the enzyme in question.

8. The method of claim 1 wherein said endohydrolase is tat-amylase.

9. A method for determining endohydrolase capable of hydrolysing polysaccharide, in aqueous samples, which comprises contacting the sample with a reagent consisting of a water-insoluble but hydrophilic, swellable, enzymat ically hydrolysable three-dimensional network of molecules of a member selected from the group consisting of the said polysaccharide and enzymatically hydrolysable derivatives thereof, said molecules being cross-linked by aliphatic bridges containing from 2 to 20 carbon atoms, with bonds of a covalent character, said network also presenting indicatable substituents bound by means of bonds of a covalent character, a reaction taking place between the enzyme and the reagent to release water-soluble fragments of the reagent containing indicatable substituents, and subsequent to the enzyme having acted upon the reagent for a determined period of time, separating undissolved reagent substances from the liquid with watersoluble fragments of the reagent dissolved therein, and then determining the indicatable substituents in at least one of the two phases consisting of the liquid phase and 9 the undissolved material phase as a measure of enzyme activity.

10. A method as claimed in claim 9, wherein the polysaccharide molecules are cross-linked by alkylene bridges containing from 3 to 20 carbon atoms. 1

11. A method as claimed in claim 10, wherein the alkylene bridges contain hydroxyl groups.

12. A method as claimed in claim 11, wherein said bridges are broken by at least' one oxygen atom.

13. A method as claimed in claim 9, wherein the reagent material is mixed with an inert material.

14. A method as claimed in claim 13, wherein said inert material is paper pulp in the form of reagent paper.

15. The method of claim 9 wherein said aliphatic bridges are selected from the group consisting of: CH .CH (H) .CH

CI-I .CH(OH) .CH- .O.CH .CH(OH) .CH OH .CH (OH) .CH .O.CH .CH .O

.CH CH (OH) .CH -CH .CH(OH) .CH .O. (CH2) 4-O- CH .CH(OH) .CH .O.CH .CH( OH) .CH .O.CH .CH(OH) .CH CH .CH (OH) .CH OH) .CH

16. The method of claim 9 wherein said endohydrolase is tit-amylase.

17. A method for determining endohydrolase capable of hydrolysing polysaccharide, in aqueous samples, which comprises contacting the sample with the reagent consisting of a water-insoluble but hydrophilic, swellable, enzymatically hydrolysable three-dimensional network of molecules of a member selected from the group consisting of the said polysaccharide and enzymatically hydrolysable derivatives thereof, said molecules being cross-linked by aliphatic bridges containing from 3 to carbon atoms, with bonds of a solvent character, said network also presenting indicatable substituents bound by means of bonds of a covalent character, a reaction taking place between v g the enzyme and the reagent to release water-soluble fragments of the reagent containing indicatable substituents, and subsequent to the enzyme having acted upon the reagent for a determined period of time, separating undissolved reagent substance from the liquid with watersoluble fragments of the reagents dissolved therein, and then determining the indicatable substituents in at least one of the two phases consisting of the liquid phase and the undissolved reagent material phase as a measure of enzyme activity.

18. A method as claimed in claim 17, wherein the polysaccharide molecules are cross-linked by alkylene bridges containing from 3 to 15 carbonatoms.

19. A method as claimed in claim 18, wherein the alkylene bridges contain hydroxyl groups.

20. A method as claimed in claim 19 wherein said bridges are broken by at least one oxygen atom.

21. A method as claimedin claim 17, wherein the reagent material-is mixed with inert material.

22. A method as claimed in claim 21, wherein the reagent material is mixed with paper pulp in the form of reagent paper.

23. The method of ,claim 17 'wher'ein said endohydrolase is u-amylase.

24. A reagentv forv determining. endohydrolase capable of hydrolysing polysaccharide, in aqueous samples, comprising a water-insoluble but hydrophilic, swellable, enzymatically hydrolysable threexlimensional network of molecules of a member selected from the group consisting of the said polysaccharide and eiizymatically hydrolysable derivatives thereof, said molecules being cross-linked by bridges with bonds of a covalent character, said network also presenting indicatable substituents bound by means of bonds of a covalent character.

25. A reagent as claimed in claim 24, when in particle form.

26. A reagent as claimed in claim 24, wherein the polysaccharide is a member selected from the group consisting of starches and dextrins thereof and enzymatically hydrolysable derivatives thereof.

27. A reagent as claimed in claim 24, wherein the indicatable substituents are colour-producing groups.

28. A reagent as claimed in claim 24, wherein the indicatable substituents are fluorescent groups.

29. A reagent as claimed in claim 24, wherein the indicatable substituents are each a member selected from the group consisting of radioactive isotopes and groups provided with radioactive isotopes.

30. A reagent for determining endohydrolase capable of hydrolysing polysaccharide, in aqueous samples, comprising a water-insoluble but hydrophilic, swellable, en- Zymatically hydrolysable three-dimentional network of molecules of a member selected from the group consisting of the said polysaccharide and enzymatically hydrolysable derivatives thereof, .said molecules being cross-linked by aliphatic bridges containing from 2 to 20 carbon atoms, with bonds of a covalent character, said network also presenting indicatable substituents bound by means of bonds of a covalent character.

31. A reagent as claimed in claim 30, wherein the polysaccharide molecules are cross-linked by alkylene bridges containing from 3 to 20 carbon atoms.

32. A reagent as claimed in claim 31, wherein the alkylene bridges contain hydroxyl groups.

33. A reagent as claimed in claim 32, wherein said bridges are broken by at least one oxygen atom.

34. A reagent as claimed in claim 30, when mixed with an inert material.

35. A reagent as claimed in claim 34, wherein said inert material is paper pulp.

36. A reagent for determining endohydrolase capable of hydrolysing polysaccharide, in aqueous samples, comprising a water-insoluble but hydrophilic, swellable, enzymatically hydrolysable three-dimensional network of molecules of a member selected from the group consisting of the said polysaccharide and enzymatically hydrolysable derivatives thereof, said molecules being crosslinked by aliphatic bridges containing from 3 to 15 carbon atoms, with bonds of a covalent character, said network also presenting indicatable substituents bound by means of bonds of a covalent character.

37. A reagent as claimed in claim 36, wherein the polysaccharide molecules are cross-linked by alkylene bridges containing from 3 to 15 carbon atoms.

38.-A reagent as claimed in claim 37, wherein the alkylene bridges contain hydroxyl groups.

39. A reagent as claimed in claim 38, wherein said bridges are broken by at elast one oxygen atom.

40. A reagent as claimed in claim 36, wherein the reagent material is mixed with an inert material.

41. A reagent as claimed in claim 40, wherein the eagent material is mixed with paper pulp.

42. The reagent of claim 36 wherein said aliphatic 11 12 OH2.CH(OH).CH(OH.CH2', OTHER REFERENCES 2-Q 2- 2- 2- 2)2 Fernley: The Use of Reactive Dyestufis in Enzymol- 2- 2- 2- z and ogy- New Substrates for Cellulolytic Enzymes. Biochem. 2- 2- 2- 2- 2)4 1., vol. 87, pp. 90-95 (196-3). 5 A. LOUIS MONACELL, Primary Examiner References Cited I. R. HOFFMAN, Assistant Examiner UNITED STATES PATENTS US. C XDR- 3,089,828 5/1963 Tsuk 195103.5 10 195 99 

